At the present time, screening tests for hepatitis C virus (HCV) and Human Immunodeficiency virus Type 1 (HIV-1) are based on the presence of antibodies to the virus in sera or plasma (Choo, Q.L., et al., Science, 244:259-262, 1989; Petricciani, J., Ann. Inter.Med., 103, 726-729, 1985). The polymerase chain reaction (PCR), an in vitro method for primer-directed enzymatic amplification of viral target sequences, has been used to detect both HCV and HIV-1 specific sequences in sera and plasma of symptomatic and asymptomatic subjects. The sensitivity of PCR allows the detection of extremely small quantities of viral genomic sequences in serum or plasma of infected individuals. Where limited volumes of clinical samples are available, a test that would allow the simultaneous detection of specific viral sequences of HCV and HIV-1 in a single step would be advantageous. Based on the nucleotide sequences of HCV (Choo, Q.L., et al., Science, 244:259-262,-1989) and the HIV-1 gag region, we have developed a specific and sensitive cDNA/polymerase chain reaction assay for the simultaneous detection of HCV and HIV-1 specific sequences in the sera of individuals. This study demonstrates that multiple primer pairs of HCV and HIV-1 can be used simultaneously in a single cDNA amplification reaction to identify specific sequences from two different viruses.